Method of treating sepsis and ARDS using chemokine alpha-2

ABSTRACT

The invention relates to the method of preventing and treating sepsis and ARDS using chemokine or biologically active fragment thereof, alone or in conjunction with an anti-infective agent.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 60/017,871 filed May 14, 1996.

FIELD OF INVENTION

[0002] This invention relates to the method of preventing and treating sepsis and adult respiratory distress syndrome using certain chemokines or biologically active fragments thereof alone or in conjunction with an anti-infective agent or hematopoietic maturing agent.

BACKGROUND OF INVENTION

[0003] Sepsis, as used herein, is broadly defined to mean situations when the invasion of a host by a microbial agent is associated with the clinical manifestations of infection including but not limited to:

[0004] (1) temperature >38° C. or <36° C.; (2) heart rate >90 beats per minute; (3) respiratory rate >20 breaths per minute or PaCO₂<32 mm Hg; (4) white blood cell count >12,000/cu mm, <4,000/cu mm, or >10% immature (band) forms; (5) organ dysfunction, hypoperfusion, or hypotension. Hypoperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, oliguria, or an acute alteration in mental states. (Chest 1992; 101: 1644-1566)

[0005] Sepsis can occur in hospitalized patients having underlying diseases or conditions that render them susceptible to bloodstream invasion or in burn, trama or surgical patents. In many cases of sepsis, the predominant pathogen is Escherichia coli, followed by other Gram-negative bacteria such as the Klebsiella-Enterobacter-Serratia group and then Pseudomonas. Although comprising a somewhat smaller percentage of infection, Gram-positive microbes such as Staphylococcus and systemic viral and fungal infections are included by the term sepsis as used herein. The genitourinary tract is the most common site of infection, the gastrointestinal tract and respiratory tract being the next most frequent sources of sepsis. Other common foci are wound, burn, and pelvic infections and infected intravenous catheters.

[0006] A serious consequence of bacterial sepsis often is septic shock. Septic shock is characterized by inadequate tissue perfusion, leading to insufficient oxygen supply to tissues, hypotension and olgiuria.

[0007] Septic shock occurs because bacterial products react with cells and components of the coagulation, complement, fibrinolytic and bradykinin systems to release proteases which injure cells and alter blood flow, especially in the capillaries.

[0008] Microorganisms frequently activate the classical complement pathway, and endotoxin activates the alternative pathway. Complement activation, leukotriene generation and the direct effects of bacterial products on neutrophils lead to accumulation of these inflammatory cells in the lungs, release of their proteolytic enzymes and toxic oxygen radicals which damage the pulmonary endothelium and initiate the adult respiratory distress syndrome (“ARDS”). ARDS is a major cause of death in patients with septic shock and is characterized by pulmonary congestion, granulocyte aggregation, haemorrhage and capillary thrombi.

[0009] Septic shock is a major cause of death in intensive care units. There are an estimated 200,000 cases per year of septic shock in the United States, and despite advances in technology (i.e., respiratory support) and antibiotic therapy, the mortality rate for septic shock remains in excess of 40%. In fact, mortality for established septic shock has decreased very little since the comprehensive description by Waisbren (Arch. Intern. Med. 88:467-488 (1951)). Although effective antibiotics are available, and there is an increased awareness of the septic shock syndrome, the incidence of septic shock over the last several decades has actually increased. With the appreciation that antimicrobial agents have failed to completely abrogate septic mortality, it is clear that other agents must be developed to be used alone or in conjunction with antimicrobials in order to rectify the deficiencies of current established therapy.

BRIEF DESCRIPTION OF THE INVENTION

[0010] This invention relates to a method of preventing or treating sepsis and ARDS comprising administering to an animal, including humans, in need thereof an effective amount of chemokine protein or biologically active fragments thereof.

[0011] This invention further relates to a method of preventing or treating sepsis and ARDS comprising administering to an animal (including humans) in need thereof an effective amount of chemokine protein or biologically active fragments thereof, either before, in conjunction with or after an anti-infective agent.

DETAILED DESCRIPTION OF THE INVENTION

[0012] It is the object of this invention to provide a new method of treatment of sepsis and ARDS comprising administering to an animal in need thereof, including humans, an effective amount of chemokine protein or biologically active fragments thereof, alone or in combination with other anti-infective agents. As used herein the term “chemokine” means those polypeptides claimed in the patent applications set forth in Table 1. TABLE 1 Chemokine Patent Applications Application Gene Name Date Filed Number Macrophage Inflammatory Protein-Gamma 22-Dec-93 08/173,209 Macrophage Inflammatory Protein-3 and -4 8-Mar-84 W095/17092 Macrophage Migration Inhibitory Factor-3 16-May-94 W095/31468 Human Chemokine Beta-9 6-Jun-95 W096/06169 Human Chemokine Polypeptides 23-Aug-94 W096/05856 Human Chemokine Beta-II and Human 8-Feb-95 US95/01780 Chemokine Alpha-1 Human Chemokine Beta-13 5-Jun-95 08/464,594 Human Chemokine Beta-12 6-Jun-95 08/468,541 Chemokine Alpha-2 19-Mar-96 60/013,653 Chemokine Alpha-3 18-Mar-96 US96/03686 Novel Chemokine for Mobilizing Stem Cells 29-Sep-95 60/006,051 Short Form Chemokine Beta-8 24-Oct-95 60/004,517

[0013] This invention further relates to a method of preventing sepsis and ARDS comprising administering to an animal in need thereof an effective amount of modified chemokine protein or biologically active fragments thereof alone or in combination with other anti-infective agents.

[0014] Known anti-infective agents include, without limitation, anti-microbial agents routinely used for the treatment of sepsis such as amino-glycosides (such as amikacin, tobramycin, netilmicin, and gentamicin), cephalosporins such as ceftazidime, related beta-lactam agents such as maxalactam, carbopenems such as imipenem, monobactam agents such as aztreonam; ampicillin and broad-spectrum penicillins, (e.g., penicillinase-resistant penicillins, ureidopenicillins or antipseudomonal penicillin or Augmentin) that are active against P. aeruginosa, Enterobacter species, indole-positive Proteus species, and Serratia. Also included within the definition of anti-infective agents are antifungal agents, amphotericin and the like as well as anti-viral agents such as famvir and acyclovir.

[0015] The compound is useful in the treatment and prevention of sepsis and ARDS in humans and other animals such as dairy cattle, horses, calves or poultry.

[0016] Chemokine protein or biologically active fragments thereof have been described in the applications in Table 1, incorporated by reference herein. The use of chemokine protein or biologically active fragments thereof for the prevention and treatment of sepsis has not been reported. It has now been discovered that chemokine protein or biologically active fragments thereof significantly increases the survival of animals challenged with lethal sepsis causing organisms. Treatment with the compound of this invention, alone or in combination with an anti-infective agent prior to contemplated thoracic or abdominal surgery would be useful in reducing the likelihood of post-operative sepsis. It may also be used post-operatively for the treatment of sepsis and ARDS caused by a variety of reasons as outlined previously.

[0017] To effectively treat a human or other animal chemokine protein or biologically active fragments thereof may be administered by injection in the dose range of about 10 fg/kg to about 100 mg/kg/dose, preferably between about 1 and 50 mg/kg/dose, or orally in the dose range of about 10 fg/kg to about 100 mg/kg body weight per dose, preferably between about 1 and 50 mg/kg body weight; if administered by infusion or similar techniques, the dose may be in the range of about 10 fg/kg to about 100 mg/kg/dose, preferably between about 1 and 50 mg/kg/dose; if administered subcutaneously the dose may be in the range of about 10 fg/kg to about 100 mg/kg/dose, preferably between about 1 and 50 mg/kg/dose.

[0018] Depending on the patient's condition, the compounds of this invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic application, the compound is administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the disease and its complications. It may be given at any time after surgery, preferably prior to 24 hours after surgery. In prophylactic applications, a composition containing chemokine protein or biologically active fragments thereof, is administered to a patient not already in a disease state to enhance the patient's resistance. It may be given one day or one week prior to surgery, preferably one to two days prior to surgery. It may be administered parenterally or orally.

[0019] Single or multiple administrations of the compounds can be carried out with dose levels and pattern being selected by the treating physician. In any event, a quantity of the compounds of the invention sufficient to effectively treat the patient should be administered.

[0020] The compounds of this invention, may also be administered in conjunction with a conventional anti-infective as disclosed herein above, such a gentamicin, augmentin or ceftazidime. The particular anti-infective chosen should be one to which the infective organism is susceptible and is selected or modified during therapy as the infecting microrganism is more particularly identified.

[0021] Additionally, various adjunctive agents in the treatment of septic shock also may be useful in combination with the components of this invention. They include sympathomimetic amines (vasopressors) such as norepinephrine, epinephrine, isoproterenol, dopamine, and dobutamine; anti-inflammatory agents such as methylprednisolone anti-inflammatory agents such as indomethacin and phenylbutazone; and corticosteroids such as betamethasone, hydrocortisone, methylprednisolone, or dexamethasone; anti-coagulants such as heparin, anti-thrombin III or coumarin type drugs for certain conditions and schedules; diuretics such as furosemide or ethacrynic acid; and antagonist of opiates and beta-endorphins such as naloxone; an antagonist of tumor necrosis factor or of interleukin-1; phenothiazines; anti-histamines; glucagon; α-adrenergic blocking agents, vasodilators; plasma expanders; packed red blood cells; platelets; cryoprecipitates; fresh frozen plasma; bacterial permeability protein; clindamycin; and antibodies to (lipid A), the J5 mutant of E. coli or to endotoxin core glycolipids. Methods for preparing such antibodies are described widely in the literature.

[0022] One of the most important aspects in the treatment of the clinical septic shock syndrome is its apparently intractable resistance to the effects of a variety of highly potent antimicrobial agents. Despite the development of newer antimicrobial agents, the overall incidence of clinical sepsis has increased, and mortality remains unacceptably high, often approaching 60% of diagnosed patients. The discovery of the increased survival with the treatment of chemokine protein or biologically active fragments thereof both prophylactically and after infection provides a new and useful therapy of sepsis and ARDS.

[0023] The compounds of this invention, may also be administered in conjunction with hematopoietic maturation agents, such as G-CSF, Flt3, M-CSF or GM-CSF. These compounds affect the mobilization of the chemokines of the invention and are believed to enhance the anti-sepsis and anti-ARDS efficacy of chemokines.

[0024] The biological activity of chemokine protein or biologically active fragments thereof are demonstrated by the following assays:

[0025] Rats. Male Fischer 344 rats obtained from Taconic farms weighing 200 to 250 g. are utilized. The rats are housed 2 per cage in standard plastic caging and fed lab chow and water ad libitum.

[0026] Chemokine protein or biologically active fragments thereof, is prepared in E. coli by the method given in Example 5. The compound is dissolved in DPBS containing 0.5% heat inactivated autologous normal rat serum. The animals are dosed intraperitoneally with chemokine 24 hours and 2 hours before infection.

[0027] Control animals are dosed with dilution buffer on the same schedule. Starting two hours after infection the rats are treated twice daily with subcutaneous gentamicin.

[0028]E. coli. A clinical isolate of E. coli isolated from sputum is utilized. The organisms are tested for antibiotic sensitivity by the disc-agar diffusion technique and found to be sensitive to gentamicin, ampicillin, cephalothin, chloramphenicol, kanamycin, tetracycline, trimethoprin/sulfamethoxazole and resistant to penicillin G, erythromycin, and vancomycin. The organism is animal passed in mice and subsequently recovered and plated onto MacConkey's agar. The reisolated organisms are grown overnight in brain-heart infusion broth, and then stored frozen at −70° C. The inoculate the fibrin clot, organisms from thawed stocks are inoculated into brainheart infusion broth and incubated overnight on a rotary shaker (120 rpm) an 37° C. The E. coli is harvested by centrifugation, washed 3× and finally resuspended in normal saline. The number or organisms is quantified by turbidimentry, and the concentration adjusted with normal saline. All inoculum sizes are based on viable counts determined by scoring colony forming units on MacConkeys agar.

[0029] Fibrin Clot. The E. coli infected fibrin clots are made from a 1% solution of bovine fibrinogen (Type 1-S, Sigma) in sterile saline. The clot is formed by adding sequentially human thrombin (Hanna Pharma.) bacteria, and fibrinogen solution to 24 well plastic plates. Bacterial numbers of 2.0 to 3.0×10⁹ are used in inoculate the fibrin clots. The resulting mixture is then incubated at room temperature for 30 minutes before implantation.

[0030] Animal Model. The rats are anethetized with ketamine/xylazine (40 mg/kg/5 mg/kg) then the abdominal surfaced is shaved and a midline laporatomy performed. Bacterial peritonitis is induced by implanting a fibrin-thrombin clot containing E. coli into the abdominal cavity. After implantation the muscle layers are closed with 4-0 silk suture, and the wound closed with surgical staples. The animals are closely observed, any animals obviously moribound are euthanized.

[0031] Gentamicin. Rats are treated subcutaneously with gentamicin sulfate (Elkins-Sinn, NJ) 5 mg/kg twice a day for five days.

[0032] Statistics. All continuously variable data are expressed as the percent survival from several pooled studies. The Fisher's Exact test is used to determine the statistical significance of the differences between the survival rates at 14 days. The differences between the groups are considered statistically significant at p<0.05.

EXAMPLE 1

[0033] Prophylactically Administered Chemokine.

[0034] Chemokine proteins may be prepared using known methods for protein purification or as described in the patent applications listed in Table 1.

[0035] The animals are dosed intraperitoneally with chemokine at doses of 10, 100 and 1,000 fg/kg, and 10 and 100 mg/kg 24 hours and 2 hours before infection. Control animals are dosed with dilution buffer on the same schedule. Starting two hours after infection the rats are treated twice daily with subcutaneous gentamicin. On day 0 the rats are implanted with an E. coli containing fibrin-thrombin clot. Starting two hours after infection the rats are treated with gentamicin twice daily. The rats prophylactically treated with chemokine at 33 or 100 fg/kg followed by gentamicin treatment demonstrated significantly improved survival rates over the diluent treated control rat receiving gentamicin therapy alone.

EXAMPLE 2

[0036] Theraputically Administered Chemokine.

[0037] On day 0 the rats are implanted with an E. coli containing fibrin-thrombin clot The animals are dosed intraperitoneally with chemokine at doses of 10, 100 and 1,000 fg/kg, and 10 and 100 mg/kg as a single injection 2 hours after infection. Control animals are dosed with dilution buffer on the same schedule. Starting two hours after infection the rats are treated twice daily with subcutaneous gentamicin. The rats theraputically treated with chemokine at 100 or 333 fg/kg followed by gentamicin treatment are assessed for improved survival rates over the diluent treated control rat receiving gentamicin therapy alone.

EXAMPLE 3

[0038] Therapeutically Administered Chemokine in S. aureus Sepsis:

[0039] On day 0 the rats are implanted with a S. aureus containing fibrin-thrombin clot. The animals are dosed intraperitoneally with chemokine at doses of 10, 100 and 1,000 fg/kg, and 10 and 100 mg/kg as a single injection 2 hours after infection. Control animals are dosed with dilution buffer on the same schedule. Starting two hours after infection the rats are treated twice daily with subcutaneous gentamicin. The rats theraputically treated with chemokine followed by gentamicin treatment are assessed for improved survival rates over the diluent treated control rat receiving gentamicin therapy alone.

1 2 1 461 DNA Homo sapiens CDS (43)..(375) 1 acgagctccg ggccgccgct ccgacgggcc agcgccctcc cc atg tcc ctg ctc 54 Met Ser Leu Leu 1 cca cgc cgc gcc cct ccg gtc agc atg agg ctc ctg gcg gcc gcg ctg 102 Pro Arg Arg Ala Pro Pro Val Ser Met Arg Leu Leu Ala Ala Ala Leu 5 10 15 20 ctc ctg ctg ctg ctg gcg ctg tac acc gcg cgt gtg gac ggg tcc aaa 150 Leu Leu Leu Leu Leu Ala Leu Tyr Thr Ala Arg Val Asp Gly Ser Lys 25 30 35 tgc aag tgc tcc cgg aag gga ccc aag atc cgc tac agc gac gtg aag 198 Cys Lys Cys Ser Arg Lys Gly Pro Lys Ile Arg Tyr Ser Asp Val Lys 40 45 50 aag ctg gaa atg aag cca aag tac ccg cac tgc gag gag aag atg gtt 246 Lys Leu Glu Met Lys Pro Lys Tyr Pro His Cys Glu Glu Lys Met Val 55 60 65 atc atc acc acc aag agc gtg tcc agg tac cga ggt cag gag cac tgc 294 Ile Ile Thr Thr Lys Ser Val Ser Arg Tyr Arg Gly Gln Glu His Cys 70 75 80 ctg cac ccc aag ctg cag agc acc aag cgc ttc atc aag tgg tac aac 342 Leu His Pro Lys Leu Gln Ser Thr Lys Arg Phe Ile Lys Trp Tyr Asn 85 90 95 100 gcc tgg aac gag aag cgc agg ttc tac gaa gaa tagggtgaaa aacctcagaa 395 Ala Trp Asn Glu Lys Arg Arg Phe Tyr Glu Glu 105 110 gggaaaactc caaaccagtt gggagacttg tggcaaagga actttgcaga ttaaaaaaaa 455 aaaaaa 461 2 111 PRT Homo sapiens 2 Met Ser Leu Leu Pro Arg Arg Ala Pro Pro Val Ser Met Arg Leu Leu 1 5 10 15 Ala Ala Ala Leu Leu Leu Leu Leu Leu Ala Leu Tyr Thr Ala Arg Val 20 25 30 Asp Gly Ser Lys Cys Lys Cys Ser Arg Lys Gly Pro Lys Ile Arg Tyr 35 40 45 Ser Asp Val Lys Lys Leu Glu Met Lys Pro Lys Tyr Pro His Cys Glu 50 55 60 Glu Lys Met Val Ile Ile Thr Thr Lys Ser Val Ser Arg Tyr Arg Gly 65 70 75 80 Gln Glu His Cys Leu His Pro Lys Leu Gln Ser Thr Lys Arg Phe Ile 85 90 95 Lys Trp Tyr Asn Ala Trp Asn Glu Lys Arg Arg Phe Tyr Glu Glu 100 105 110 

What is claimed is:
 1. A method of treating sepsis and ARDS comprising administering to an animal in need thereof an effective amount of chemokine selected from the group consisting of: (a) Macrophage Inflammatory Protein-Gamma; (b) Macrophage Inflammatory Protein-3 and-4; (c) Macrophage Migration Inhibitory Factor-3; (d) Human Chemokine Beta-9; (e) Human Chemokine Polypeptides; (f) Human Chemokine Beta-11 and Human Chemokine Alpha-1; (g) Human Chemokine Beta-13; (h) Human Chemokine Beta-12; (i) Chemokine Alpha-2; (j) chemokine Alpha-3; (k) Novel Chemokine for Mobilizing Stem Cells; (l) Short Form Chemokine Beta-8; or biologically active fragments thereof.
 2. A method according to claim 1 wherein said effective amount is from about 1 to about 100 mg/kg/dose.
 3. The method according to claim 1 wherein chemokine is administered orally.
 4. The method according to claim 1 wherein chemokine is administered subcutaneously.
 5. A method of treating sepsis and ARDS comprising administering to an animal in need thereof an effective amount of chemokine or biologically active fragments thereof in conjunction with an effective amount of an anti-infective agent.
 6. A method according to claim 5 wherein the anti-infective agent is selected from the group consisting of gentamicin, augmentin or ceftazidime.
 7. A method for the prevention of sepsis and ARDS comprising administering to an animal in need thereof an effective amount of chemokine or biologically active fragment thereof.
 8. A method according to claim 7 wherein the effective amount is from about 1 to about 100 mg/kg/dose.
 9. The method according to claim 7 wherein cheomkine or biologically active fragment thereof is administered 1 to 2 days prior to surgery.
 10. A method for the prevention of sepsis and ARDS comprising administering to an animal in need thereof an effective amount of chemokine or biologically active fragment thereof, in conjunction with an effective amount of an anti-infective agent.
 11. A method according to claim 10 wherein the anti-infective agent is selected from the group consisting of gentamicin, augmentin or ceftazidime.
 12. A method for the treatment of sepsis and ARDS comprising administering to an animal in need thereof an effective amount of chemokine or biologically active fragment thereof.
 13. A method according to claim 12 wherein the effective amount is from about 1 to about 100 mg/kg/dose.
 14. The method according to claim 12 wherein chemokine or biologically active fragment thereof is administered 2 hours to 24 hours after surgery.
 15. A method for the treatment of sepsis and ARDS comprising administering to an animal in need the an effective amount of chemokine or biologically active fragment thereof, in conjunction with an effective amount of an anti-infective agent.
 16. A method according to claim 15 wherein the anti-infective agent is selected from the group consisting of gentamicin, augmentin or ceftazidime. 